Each year, there are approximately 200,000 craniofacial fractures requiring bone transplantation in the US with an economic burden of $2B. These injuries often require multiple complex surgeries, which do not achieve adequate functional or aesthetic restoration. To address this limitation, the field of tissue engineering has employed advanced approaches that combine a patient?s own cells with customized bioactive scaffolds to induce regeneration. For efficacious clinical translation of tissue engineering strategies, it is crucial to develop them as point-of-care technologies in which the harvesting of cells, their packaging into scaffolds, and immediate transplantation into the defect site will take place within a single surgical procedure. A major hurdle of this strategy is that the hypoxic wound microenvironment impedes the ability of surviving cells to orchestrate regeneration. To overcome this limitation, we propose to design scaffolds capable of delivering oxygen (O2) along with the cells. Specifically, we will embed O2-eluting microtanks (tanks) ? hollow, polymeric microspheres capable of ?storing? O2 at elevated pressures and slowly releasing it into the cellular microenvironment ? into scaffolds comprised of polycaprolactone (PCL) and decellularized bone matrix (DCB) that are 3D-printed in precise, anatomic shapes. To effectively design O2-eluting, PCL-DCB-tank scaffolds and track the enhanced viability and therapeutic efficacy of transplanted stromal vascular fraction (SVF) cells harvested from lipoaspirate, we will utilize multimodal in vivo optical imaging. This will provide quantitative data on the in vivo microenvironmental factors that impact stem cell survival and tissue regeneration following transplantation and uniquely inform the design process leading to more effective, next-generation biomaterial scaffolds. We hypothesize that the delivery of oxygen using our microtank technology for up to four days will enhance stem cell survival, vascularization and bone formation within the defect and that by non-invasively monitoring the effects of oxygen delivery via a cranial window, we can optimize the design of the scaffold. In Specific Aim 1, we will manufacture 10-50 m diameter biodegradable polyvinyl alcohol microtanks, incorporate them into the struts of the 3D-printed scaffolds, and validate the spatiotemporal O2 gradients within the scaffolds in response to varying the microtank concentrations and loading pressures. In Specific Aim 2, we will integrate experimental data of O2 concentrations most favorable to vascular morphogenesis/osteogenic differentiation of SVF with numerical simulations to predict the scaffold designs that provide favorable spatiotemporal O2 gradients to promote tissue regeneration. In Specific Aim 3, we will utilize non-invasive, multimodal imaging to dynamically monitor transplanted cells and vascular assembly in PCL-DCB-tank scaffolds and use this to enhance scaffold design. We will test the optimal designs in a scaled-up, stringent, vasculature-limited model of bone regeneration. The complementary tissue engineering/imaging strengths will provide unprecedented insight into bone regeneration and produce novel platform biomaterial technologies.